Recently the signaling enzyme, sphingosine kinase, has been cloned and receptors for its product, sphingosine-1-phosphate, have been identified, Consequently, the central role of sphingosine-kinase in several important signal transduction pathways has emerged. Sphingosine kinase is activated by a number of biologically important agonists to promote cell proliferation, cell survival, and migration, as well as other cellular responses. Importantly, the activation of sphingosine-kinase results in targeting of the enzyme to cell membranes from the cytosol. The exact sites to which the activated enzyme is targeted are unknown. Translocation of signaling enzymes is now recognized as a critical feature controlling the strength and specificity of many signal transduction pathways. The hypothesis guiding the research outlined in this proposal is that the targeting of agonist-stimulated sphingosine kinase to distinct intracellular sites is critical to the signaling function of this enzyme. The goal of this proposal is to determine how translocation of sphingosine kinase affects the localized production and distribution of the lipid second messenger sphingosine-1-phosphate, and how this distribution affects signaling function, particularly in generation of the transformed phenotype. An important part of this study will be to establish a structural basis of the interaction of sphingosine-kinase with its substrates and with biological membranes in order to understand the functional consequences of translocation in detail. To accomplish these goals the following specific aims are proposed: Aim 1. Test the hypothesis that activation leads to targeting of sphingosine-kinase and consequently sphingosine-1-phosphate, to specific sites within the cell. Aim II.Test the hypothesis that the compartmentalization of activated sphingosine-kinase is critical for signaling function in proliferation and transformation. Aim III. Determine the structural basis of sphingosine-kinase signalling. We have recently shown that overexpression of sphingosine-kinase results in cellular transformation and tumorogenesis. This indicates that sphingosine-kinase controls important pathways that may lead to cancers. Our long-term goal is to capitalize on the understanding of these pathways to discover novel therapies impacting on human cancers.